1-oxo-and 1,3-dioxoisoindolines

ABSTRACT

1-Oxo- and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines substituted in the 4- and/or 7-position of the isoindoline ring and optionally further substituted in the 3-position of the 2,6-dioxopiperidine ring reduce the levels of inflammatory cytokines such as TNFα in a mammal. A typical embodiment is 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/270,411 filed Mar. 16, 1999, currently pending, which claims thebenefit of U.S. Provisional Application No. 60/078,180 filed on Mar. 16,1998 entitled 1-Oxo- and 1,3-Dioxoisoindolines and Method of ReducingInflammatory Cytokine Levels, hereby incorporated by reference into thisapplication.

BACKGROUND OF THE INVENTION

Tumor necrosis factor-α, or TNFα, is a cytokine which is releasedprimarily by mononuclear phagocytes in response to a numberimmunostimulators. It is a key proinflammatory cytokine in theinflammation cascade causing the production and/or release of othercytokines and agents. When administered to animals or humans, it causesinflammation, fever, cardiovascular effects, hemorrhage, coagulation,and acute phase responses similar to those seen during acute infectionsand shock states. Excessive or unregulated TNFα production thus has beenimplicated in a number of disease conditions. These include endotoxemiaand/or toxic shock syndrome {Tracey et al., Nature 330, 662-664 (1987)and Hinshaw et al., Circ. Shock 30, 279-292 (1990)}; cachexia {Dezube etal., Lancet, 335 (8690), 662 (1990)} and Adult Respiratory DistressSyndrome (ARDS) where TNFα concentration in excess of 12,000 pg/mL havebeen detected in pulmonary aspirates from ARDS patients {Millar et al.,Lancet 2(8665), 712-714 (1989)}. Systemic infusion of recombinant TNFαalso resulted in changes typically seen in ARDS {Ferrai-Baliviera etal., Arch. Surg. 124(12), 1400-1405 (1989)}.

TNFα appears to be involved in bone resorption diseases, includingarthritis. When activated, leukocytes will produce bone-resorption, anactivity to which the data suggest TNFα contributes. {Bertolini et al.,Nature 319, 516-518 (1986) and Johnson et al, Endocrinology 124(3),1424-1427 (1989).} TNFα also has been shown to stimulate bone resorptionand inhibit bone formation in vitro and in vivo through stimulation ofosteoclast formation and activation combined with inhibition ofosteoblast function. Although TNFα may be involved in many boneresorption diseases, including arthritis, the most compelling link withdisease is the association between production of TNFα by tumor or hosttissues and malignancy associated hypercalcemia {Calci. Tissue Int. (US)46(Suppl.), S3-10 (1990)}. In Graft versus Host Reaction, increasedserum TNFα levels have been associated with major complication followingacute allogenic bone marrow transplants {Holler et al., Blood, 75(4),1011-1016 (1990)}.

Cerebral malaria is a lethal hyperacute neurological syndrome associatedwith high blood levels of TNFα and the most severe complicationoccurring in malaria patients. Levels of serum TNFα correlated directlywith the severity of disease and the prognosis in patients with acutemalaria attacks {Grau et al., N. Engl. J. Med. 320(24), 1586-1591(1989)}.

Macrophage-induced angiogenesis is known to be mediated by TNFα.Leibovich et al. {Nature, 329, 630-632 (1987)} showed TNFα induces invivo capillary blood vessel formation in the rat cornea and thedeveloping chick chorioallantoic membranes at very low doses and suggestTNFα is a candidate for inducing angiogenesis in inflammation, woundrepair, and tumor growth. TNFα production also has been associated withcancerous conditions, particularly induced tumors {Ching et al., Brit.J. Cancer, (1955) 72, 339-343, and Koch, Progress in MedicinalChemistry, 22, 166-242 (1985)}.

TNFα also plays a role in the area of chronic pulmonary inflammatorydiseases. The deposition of silica particles leads to silicosis, adisease of progressive respiratory failure caused by a fibroticreaction. Antibody to TNFα completely blocked the silica-induced lungfibrosis in mice {Pignet et al., Nature, 344, 245-247 (1990)}. Highlevels of TNFα production (in the serum and in isolated macrophages)have been demonstrated in animal models of silica and asbestos inducedfibrosis {Bissonnette et al., Inflammation 13(3), 329-339 (1989)}.Alveolar macrophages from pulmonary sarcoidosis patients have also beenfound to spontaneously release massive quantities of TNFα as comparedwith macrophages from normal donors {Baughman et al., J. Lab. Clin. Med.115(1), 36-42 (1990)}.

TNFα is also implicated in the inflammatory response which followsreperfusion, called reperfusion injury, and is a major cause of tissuedamage after loss of blood flow {Vedder et al., PNAS 87, 2643-2646(1990)}. TNFα also alters the properties of endothelial cells and hasvarious pro-coagulant activities, such as producing an increase intissue factor pro-coagulant activity and suppression of theanticoagulant protein C pathway as well as down-regulating theexpression of thrombomodulin {Sherry et al., J. Cell Biol. 107,1269-1277 (1988)}. TNFα has pro-inflammatory activities which togetherwith its early production (during the initial stage of an inflammatoryevent) make it a likely mediator of tissue injury in several importantdisorders including but not limited to, myocardial infarction, strokeand circulatory shock. Of specific importance may be TNFα-inducedexpression of adhesion molecules, such as intercellular adhesionmolecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) onendothelial cells {Munro et al., Am. J. Path. 135(1), 121-132 (1989)}.

TNFα blockage with monoclonal anti-TNFα antibodies has been shown to bebeneficial in rheumatoid arthritis {Elliot et al., Int. J. Pharmac. 199517(2), 141-145}. High levels of TNFα are associated with Crohn's disease{von Dullemen et al., Gastroenterology, 1995 109(1), 129-135} andclinical benefit has been achieved with TNFα antibody treatment.

Moreover, it now is known that TNFα is a potent activator of retrovirusreplication including activation of HIV-1. {Duh et al., Proc. Nat. Acad.Sci. 86, 5974-5978 (1989); Poll et al., Proc. Nat. Acad. Sci. 87,782-785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., J.Immunol. 142, 431-438 (1989); Poll et al., AIDS Res. Hum. Retrovirus,191-197 (1992)}. AIDS results from the infection of T lymphocytes withHuman Immunodeficiency Virus (HIV). At least three types or strains ofHIV have been identified, i.e., HIV-1, HIV-2 and HIV-3. As a consequenceof HIV infection, T-cell mediated immunity is impaired and infectedindividuals manifest severe opportunistic infections and/or unusualneoplasms. HIV entry into the T lymphocyte requires T lymphocyteactivation. Other viruses, such as HIV-1, HIV-2 infect T lymphocytesafter T cell activation and such virus protein expression and/orreplication is mediated or maintained by such T cell activation. Once anactivated T lymphocyte is infected with HIV, the T lymphocyte mustcontinue to be maintained in an activated state to permit HIV geneexpression and/or HIV replication. Cytokines, specifically TNFα, areimplicated in activated T-cell mediated HIV protein expression and/orvirus replication by playing a role in maintaining T lymphocyteactivation. Therefore, interference with cytokine activity such as byprevention or inhibition of cytokine production, notably TNFα, in anHIV-infected individual assists in limiting the maintenance of Tlymphocyte caused by HIV infection.

Monocytes, macrophages, and related cells, such as kupffer and glialcells, also have been implicated in maintenance of the HIV infection.These cells, like T cells, are targets for viral replication and thelevel of viral replication is dependent upon the activation state of thecells. {Rosenberg et al., The Immunopathogenesis of HIV Infection,Advances in Immunology, 57 (1989)}. Cytokines, such as TNFα, have beenshown to activate HIV replication in monocytes and/or macrophages {Poliet al., Proc. Natl. Acad. Sci., 87, 782-784 (1990)}, therefore,prevention or inhibition of cytokine production or activity aids inlimiting HIV progression for T cells. Additional studies have identifiedTNFα as a common factor in the activation of HIV in vitro and hasprovided a clear mechanism of action via a nuclear regulatory proteinfound in the cytoplasm of cells (Osborn, et al., PNAS 86 2336-2340).This evidence suggests that a reduction of TNFα synthesis may have anantiviral effect in HIV infections, by reducing the transcription andthus virus production.

AIDS viral replication of latent HIV in T cell and macrophage lines canbe induced by TNFα {Folks et al., PNAS 86, 2365-2368 (1989)}. Amolecular mechanism for the virus inducing activity is suggested byTNFα's ability to activate a gene regulatory protein (NFκB) found in thecytoplasm of cells, which promotes HIV replication through binding to aviral regulatory gene sequence (LTR) {Osborn et al., PNAS 86, 2336-2340(1989)}. TNFα in AIDS associated cachexia is suggested by elevated serumTNFα and high levels of spontaneous TNFα production in peripheral bloodmonocytes from patients {Wright et al., J. Immunol. 141(1), 99-104(1988)}. TNFα has been implicated in various roles with other viralinfections, such as the cytomegalia virus (CMV), influenza virus,adenovirus, and the herpes family of viruses for similar reasons asthose noted.

The nuclear factor κB (NFκB) is a pleiotropic transcriptional activator(Lenardo, et al., Cell 1989, 58, 227-29). NFκB has been implicated as atranscriptional activator in a variety of disease and inflammatorystates and is thought to regulate cytokine levels including but notlimited to TNFα and also to be an activator of HIV transcription(Dbaibo, et al., J. Biol. Chem. 1993, 17762-66; Duh et al., Proc. Natl.Acad. Sci. 1989, 86, 5974-78; Bachelerie et al., Nature 1991, 350,709-12; Boswas et al., J. Acquired Immune Deficiency Syndrome 1993, 6,778-786; Suzuki et al., Biochem. And Biophys. Res. Comm. 1993, 193,277-83; Suzuki et al., Biochem. And Biophys. Res Comm. 1992, 189,1709-15; Suzuki et al., Biochem. Mol. Bio. Int. 1993, 31(4), 693-700;Shakhov et al., Proc. Natl. Acad. Sci. USA 1990, 171, 35-47; and Staalet al., Proc. Natl. Acad. Sci. USA 1990, 87, 9943-47). Thus, inhibitionof NFκB binding can regulate transcription of cytokine gene(s) andthrough this modulation and other mechanisms be useful in the inhibitionof a multitude of disease states. The compounds described herein caninhibit the action of NFκB in the nucleus and thus are useful in thetreatment of a variety of diseases including but not limited torheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, otherarthritic conditions, septic shock, septis, endotoxic shock, graftversus host disease, wasting, Crohn's disease, ulcerative colitis,multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV,AIDS, and opportunistic infections in AIDS. TNFα and NFκB levels areinfluenced by a reciprocal feedback loop. As noted above, the compoundsof the present invention affect the levels of both TNFα and NFκB.

Many cellular functions are mediated by levels of adenosine 3′,5′-cyclicmonophosphate (cAMP). Such cellular functions can contribute toinflammatory conditions and diseases including asthma, inflammation, andother conditions (Lowe and Cheng, Drugs of the Future, 17(9), 799-807,1992). It has been shown that the elevation of cAMP in inflammatoryleukocytes inhibits their activation and the subsequent release ofinflammatory mediators, including TNFα and NFκB. Increased levels ofcAMP also leads to the relaxation of airway smooth muscle.Phosphodiesterases control the level of cAMP through hydrolysis andinhibitors of phosphodiesterases have been shown to increase cAMPlevels.

Decreasing TNFα levels and/or increasing cAMP levels thus constitutes avaluable therapeutic strategy for the treatment of many inflammatory,infectious, immunological or malignant diseases. These include but arenot restricted to septic shock, sepsis, endotoxic shock, hemodynamicshock and sepsis syndrome, post ischemic reperfusion injury, malaria,mycobacterial infection, meningitis, psoriasis, congestive heartfailure, fibrotic disease, cachexia, graft rejection, cancer, autoimmunedisease, opportunistic infections in AIDS, rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, other arthritic conditions,Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupuserythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolarinjury. Prior efforts directed to the suppression of the effects of TNFαhave ranged from the utilization of steroids such as dexamethasone andprednisolone to the use of both polyclonal and monoclonal antibodies{Beutler et al., Science 234,470-474 (1985); WO 92/11383}.

DETAILED DESCRIPTION

The present invention is based on the discovery that certain classes ofnon-polypeptide compounds more fully described herein decrease thelevels of TNFα, increase cAMP levels, and inhibit inflammatorycytokines. The present invention thus relates to 1-oxo- and1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines substituted in the4-position of the isoindoline ring and optionally further substituted inthe 3-position of the 2,6-dioxopiperidine ring, the method of reducinglevels of tumor necrosis factor α and other inflammatory cytokines in amammal through the administration of such derivatives, andpharmaceutical compositions containing such derivatives.

In particular, the invention pertains to

(a) a 2-(2,6-dioxopiperidin-3-yl)-isoindoline of the formula:

 in which

Y is oxygen or H₂,

a first of R¹ and R² is halo, alkyl, alkoxy, alkylamino, dialkylamino,cyano, or carbamoyl, the second of R¹ and R², independently of thefirst, is hydrogen, halo, alkyl, alkoxy, alkylamino, dialkylamino,cyano, or carbamoyl, and

R³ is hydrogen, alkyl, or benzyl, and

(b) the acid addition salts of said2-(2,6-dioxopiperidin-3-yl)-isoindolines which contain a nitrogen atomcapable of being protonated.

Unless otherwise defined, the term alkyl denotes a univalent saturatedbranched or straight hydrocarbon chain containing from 1 to 4 carbonatoms. Representative of such alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl. Alkoxy refers toan alkyl group bound to the remainder of the molecule through anethereal oxygen atom. Representative of such alkoxy groups are methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, andtert-butoxy.

Halo includes bromo, chloro, fluoro, and iodo.

The compounds of Formula I are used, under the supervision of qualifiedprofessionals, to inhibit the undesirable effects of TNFα and otherinflammatory cytokines including the interleukins IL-1, IL-6, and IL-12.The compounds can be administered orally, rectally, or parenterally,alone or in combination with other therapeutic agents includingantibiotics, steroids, chemotherapeutic agents, etc., to a mammal inneed of treatment; e.g., in the treatment of cancers, rheumatoidarthritis, inflammatory bowel disease, muscular dystrophy, Crohn'sdisease, etc.

The compounds of the present invention also can be used topically in thetreatment or prophylaxis of disease states mediated or exacerbated byexcessive TNFα production, respectively, such as viral infections, suchas those caused by the herpes viruses, or viral conjunctivitis,psoriasis, atopic dermatitis, etc.

The compounds also can be used in the veterinary treatment of mammalsother than humans in need of prevention or inhibition of TNFαproduction. TNFα mediated diseases for treatment, therapeutically orprophylactically, in animals include disease states such as those notedabove, but in particular viral infections. Examples include felineimmunodeficiency virus, equine infectious anaemia virus, caprinearthritis virus, visna virus, and maedi virus, as well as otherlentiviruses.

The compounds of Formula I are readily prepared through a number ofroutes. In a first embodiment, an anhydride or lactone is allowed toreact with a 3-amino-2,6-dioxo-piperidine:

In the foregoing reactions, each of R¹, R², R³, and Y are as definedabove.

The 3-amino-2,6-dioxopiperidine can be obtained from the correspondingglutamic acid anhydride through conventional amidation or from thecyclization of appropriate glutamine derivatives.

The compounds in which Y is H₂ alternatively can be obtained from adisubstituted benzoate intermediate according to the followingreactions:

in which R⁴ is CHO or CH₂Br in the presence of an acid acceptor such asdimethylaminopyridine or triethylamine.

The disubstituted benzoate intermediates are known or can be obtainedthough conventional processes. For example, a lower alkyl ester of a3,6-disubstituted ortho-toluic acid is brominated withN-bromosuccinimide under the influence of light to yield the lower alkyl2-(bromomethyl)-3,6-disubstitutedbenzoate.

Alternatively, a dialdehyde is allowed to react with2,6-dioxopiperidin-3-ammonium chloride to obtain the compounds ofFormula I in which Y is H₂:

Finally, a dialdehyde is allowed to react with glutamine and theresulting 2-(1-oxo-isoindolin-2-yl)glutaric acid then cyclized to yielda 4,7-disubstituted 1-oxo-2-(2,6-dioxo-piperidin-3-yl)-isoindoline ofFormula I in which Y is H₂:

The carbon atom to which R³ is bound in the compounds of Formula Iconstitutes a center of chirality, thereby giving rise to opticalisomers:

Both the racemates of these isomers and the individual isomersthemselves, as well as diastereomers when a second chiral center ispresent, are within the scope of the present invention. The racematescan be used as such or can be separated into their individual isomersmechanically as by chromatography using a chiral absorbent.Alternatively, the individual isomers can be prepared in chiral form orseparated chemically from a mixture by forming salts with a chiral acidor base, such as the individual enantiomers of 10-camphorsulfonic acid,camphoric acid, α-bromocamphoric acid, methoxyacetic acid, tartaricacid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid,and the like, and then freeing one or both of the resolved bases,optionally repeating the process, so as obtain either or bothsubstantially free of the other; i.e., in a form having an opticalpurity of >95%.

The present invention also pertains to the physiologically acceptablenon-toxic acid addition salts of the compound of Formula I which containa group capable of being protonated; e.g., amino. Such salts includethose derived from organic and inorganic acids such as, withoutlimitation, hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid, methanesulphonic acid, acetic acid, tartaric acid, lacticacid, succinic acid, citric acid, malic acid, maleic acid, sorbic acid,aconitic acid, salicylic acid, phthalic acid, embonic acid, enanthicacid, and the like.

Particularly preferred compounds include1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-ethylisoindoline,1,3-dioxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4-methylisoindoline,1,3-dioxo-2-(2,6-dioxoopiperidin-3-yl)-4,7-dimethylisoindoline,1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4-ethylisoindoline,1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4-methylisoindoline,1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-7-ethylisoindoline,1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-7-methylisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-propylisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-chloroisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-carbamoylisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methoxyisoindoline,1-oxo-2-(2,6-dioxoopiperidin-3-yl)-4,7-dimethylisoindoline,1-oxo-2-(2,6-dioxoopiperidin-3-yl)-4-methyl-7-ethylisoindoline, and1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,7-diethoxyisoindoline. Of these,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4,7-dimethylisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline, and 1oxo-2-(2,6-dioxopiperidin-3-yl)-4,7-dimethylisoindoline are particularlypreferred.

Oral dosage forms include tablets, capsules, dragees, and similarshaped, compressed pharmaceutical forms containing from 1 to 100 mg ofdrug per unit dosage. Isotonic saline solutions containing from 20 to100 mg/mL can be used for parenteral administration which includesintramuscular, intrathecal, intravenous and intra-arterial routes ofadministration. Rectal administration can be effected through the use ofsuppositories formulated from conventional carriers such as cocoabutter.

Pharmaceutical compositions thus comprise one or more compounds ofFormulas I associated with at least one pharmaceutically acceptablecarrier, diluent or excipient. In preparing such compositions, theactive ingredients are usually mixed with or diluted by an excipient orenclosed within such a carrier which can be in the form of a capsule orsachet. When the excipient serves as a diluent, it may be a solid,semi-solid, or liquid material which acts as a vehicle, carrier, ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, elixirs, suspensions, emulsions,solutions, syrups, soft and hard gelatin capsules, suppositories,sterile injectable solutions and sterile packaged powders. Examples ofsuitable excipients include lactose, dextrose, sucrose, sorbitol,mannitol, starch, gum acacia, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidinone, cellulose, water, syrup, and methylcellulose, the formulations can additionally include lubricating agentssuch as talc, magnesium stearate and mineral oil, wetting agents,emulsifying and suspending agents, preserving agents such as methyl- andpropylhydroxybenzoates, sweetening agents or flavoring agents.

The compositions preferably are formulated in unit dosage form, meaningphysically discrete units suitable as a unitary dosage, or apredetermined fraction of a unitary dose to be administered in a singleor multiple dosage regimen to human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with a suitablepharmaceutical excipient. The compositions can be formulated so as toprovide an immediate, sustained or delayed release of active ingredientafter administration to the patient by employing procedures well knownin the art.

Enzyme-linked immunosorbent assays for TNFα can be performed in aconventional manner. PBMC is isolated from normal donors byFicoll-Hypaque density centrifugation. Cells are cultured in RPMIsupplemented with 10% AB+ serum, 2mM L-glutamine, 100 U/mL penicillin,and 100 mg/mL streptomycin. Drugs are dissolved in dimethylsulfoxide(Sigma Chemical) and further dilutions are done in supplemented RPMI.The final dimethylsulfoxide concentration in the presence or absence ofdrug in the PBMC suspensions is 0.25 wt %. Drugs are assayed at half-logdilutions starting at 50 mg/mL. Drugs are added to PBMC (10⁶ cells/mL)in 96 wells plates one hour before the addition of LPS. PBMC (10⁶cells/mL) in the presence or absence of drug are stimulated by treatmentwith 1 mg/mL of LPS from Salmonella minnesota R595 (List BiologicalLabs, Campbell, Calif.). Cells are then incubated at 37° C. for 18-20hours. Supernatants are harvested and assayed immediately for TNFαlevels or kept frozen at −70° C. (for not more than 4 days) untilassayed. The concentration of TNFα in the supernatant is determined byhuman TNFα ELISA kits (ENDOGEN, Boston, Mass.) according to themanufacturer's directions.

The following examples will serve to further typify the nature of thisinvention but should not be construed as a limitation in the scopethereof, which scope is defined solely by the appended claims.

EXAMPLE 1 2-(2,6-Dioxopiperid-3-yl)-4-methylisoindoline-1,3-dione

A stirred solution of 3-methylphthalic anhydride (2.96 g, 18.2 mmol),3-aminopiperidine-2,6-dione hydrogen chloride (3.00 g, 18.2 mmol) andsodium acetate (1.57 g, 19.1 mmol) in acetic acid (30 mL) was heated atreflux for 23 hours. The solvent was removed in vacuo to give a solidwhich was stirred with water (40 mL) for 1 hour, filtered, washed withwater (30 mL), and then heated with decolorizing charcoal (1 g) inacetone (2 L) at reflux temperature for 30 min. The suspension wasfiltered through a pad of Celite to give a clear solution. The solventof filtrate was removed in vacuo to give2-(2,6-dioxopiperid-3-yl)-4-methylisoindoline-1,3-dione as a white solid(4.08 g, 82% yield); mp 290.0-292.0° C.; ¹H NMR (DMSO-d6); δ2.03-2.09(m, 1H, CHH), 2.50-2.60 (m, 2H, CH₂), 2.63 (s, 3H, CH₃), 2.83-2.95 (m,IH, CHH), 5.13 (dd, J=5.4,12.3 Hz, IH, NCH), 7.65-7.79 (m, 3H, Ar),11.13 (br s, IH, NH); ¹³C NMR (DMSO-d6) δ17.04, 21.99, 30.93, 48.76,121.05, 127.89, 131.63, 134.37, 136.91, 137.61, 167.04, 167.83, 169.87,172.74; Anal Calcd for C₁₄H₁₂N₂O₄: C, 61.76; H, 4.44; N, 10.29. Found:C, 61.68; H, 4.37; N, 10.17.

EXAMPLE 2

By substituting equivalent amounts of 3-ethylphthalic anhydride,3-fluorophthalic anhydride, 3-chlorophthalic anhydride,3-carbamoylphthalic anhydride, and 3-methoxyphthalic anhydride in theprocedure of Example 1, there are respectively obtained1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-ethylisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-chloroisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-carbamoylisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methoxyisoindoline.

EXAMPLE 3

By substituting equivalent amounts of3-amino-3-methylpiperidine2,6-dione hydrogen chloride for3-aminopiperidine2,6-dione hydrogen chloride in the procedure of Example1, 1,3-dioxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4-methylisoindoline isobtained.

EXAMPLE 4 1-Oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline

A mixture of 16.25 g of 2,6-dioxopiperidin-3-ammonium chloride, and 30.1g of methyl 2-bromomethyl-3-methylbenzoate, and 12.5 g of triethylaminein 100 mL of dimethylformamide is stirred at room temperature for 15hours. The mixture is then concentrated in vacuo and the residue mixedwith methylene chloride and water. The aqueous layer is separated andback-extracted with methylene chloride. The combined methylene chloridesolutions are dried over magnesium sulfate and concentrated in vacuo togive 1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.

In a similar fashion1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,7-dimethylisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-ethylisoindoline, and1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methoxyisoindoline are obtained bysubstituting equivalent amounts of methyl2-bromomethyl-3,6-dimethylbenzoate, methyl2-bromomethyl-3-ethylbenzoate, and methyl2-bromomethyl-3-methoxybenzoate, respectively, for methyl2-bromomethyl-3-methylbenzoate.

EXAMPLE 5 2-(2,6-Dioxopiperidin-3-yl)-4,7-dimethylisoindoline-1,3-dione

2-(2,6-Dioxopiperid-3-yl)-4,7-dimethylisoindoline-1,3-dione was preparedby the procedure of Example 1 from 3,6-dimethylphthalic anhydride (220mg, 1.25 mmol), 3-aminopiperidine-2,6-dione hydrogen chloride (204 mg,1.24 mmol) and sodium acetate (110 mg, 1.34 mmol) in acetic acid (10mL). The product is a white solid (200 mg, 56% yield): mp 263.0-265.0°C.; ¹H NMR (DMSO-d₆) δ2.01-2.07 (m, 1H, CHH), 2.50-2.89 (m, 9H, CH₃,CHH, CH₂), 5.10 (dd, J=5.1, 12.4 Hz, 1H, NCH), 7.52 (s, 2H, Ar), 11.12(br s, 1H, NH); ¹³C NMR (DMSO-d₆) δ16.82, 22.02, 30.97, 48.59, 128.01,135.04, 136.58, 167.68, 169.98, 172.83.

EXAMPLE 6 2-(2,6-Dioxo(3-piperidyl))-4-ethylisoindoline-1,3-dione

2-(2,6-Dioxo(3-piperidyl))-4-ethylisoindoline-1,3-dione was prepared bythe procedure of Example 1 from 3-ethylphthalic anhydride (0.860 g, 4.89mmol), 3-aminopiperidine-2,6-dione hydrogen chloride (0.803 g, 4.88mmol) and sodium acetate (0.420 g, 5.12 mmol) in acetic acid (10 mL).The product was a white solid (1.06 g, 76% yield); mp, 235.0-236.5° C.;¹H NMR (DMSO-d₆) δ1.22 (t, J=7.4 Hz, 3H, CH₃), 2.04-2.10 (m, 1H, CHH),2.47-2.63 (m, 2H, CH₂), 2.83-2.98 (m, 1H, CHH), 3.07 (q, J=7.5 Hz, 2H,CH₂), 5.13 (dd, J=5.4, 12.5 Hz, 1H, NCH), 7.70-7.82 (m, 3H, Ar), 11.13(br s, 1H, NH); ¹³C NMR (DMSO-d₆) δ14.84, 21.95, 23.69, 30.90, 48.77,121.09, 127.26, 131.76, 134.63, 135.39, 143.87, 166.99, 167.58, 169.85,172.72; Anal Calcd for C₁₅H₁₄N₂O₄: C, 62.93; H, 4.93; N, 9.79. Found: C,62.74; H, 4.84; N, 9.54.

EXAMPLE 7 4-Methoxy-2-(2,6-dioxo(3-piperidyl))isoindoline-1,3-dione

4-Methoxy-2-(2,6-dioxo(3-piperidyl))isoindoline-1,3-dione was preparedby the procedure of Example 1 from 3-methoxyphthalic anhydride (1.0 g,5.6 mmol) {Rao. A. V. R. et al, Indian J. Chem. 1981, 20 (B), 248},3-aminopiperidine-2,6-dione hydrogen chloride (0.92 g, 5.6 mmol) andsodium acetate (0.48 g, 6.0 mmol) in acetic acid (20 mL). The productwas a white solid (0.44 g, 27% yield); mp, 281.5-282.5° C.; ¹H NMR(DMSO-d₆) δ2.00-2.08 (m, 1H, CHH), 2.56-2.62 (m, 2H, CH₂), 2.82-2.91 (m,1H, CHH), 3.97 (s, 3H, CH₃), 5.08 (dd, J=5.3, 12.8 Hz, 1H, NCH), 7.46(d, J=7.2 Hz, 1H, Ar), 7.52 (d, J=8.5 Hz, 1H, Ar), 7.84 (d, J=7.8 Hz,1H, Ar), 11.10 (br s, 1H, NH); ¹³C NMR (DMSO-d₆) δ21.97, 30.92, 48.73,56.33, 115.24, 116.11, 119.01, 133.19, 137.15, 156.49, 165.37, 166.84,169.94, 172.79; Anal Calcd for C₁₄H₁₂N₂O₅: C, 58.33; H, 4.20; N, 9.72.Found: C, 58.23; H, 3.90; N, 9.53.

EXAMPLE 84-Dimethylamino-2-(2,6-dioxo(3-piperidyl))isoindoline-1,3-dione

4-Dimethylamino-2-(2,6-dioxo(3-piperidyl))isoindoline-1,3-dione wasprepared by the procedure of Example 1 from 3-dimethylaminophthalicanhydride (1.34 g, 7.0 mmol), 3-aminopiperidine-2,6-dione hydrogenchloride (1.15 g, 7.0 mmol) and sodium acetate (0.60 g, 7.3 mmol) inacetic acid (20 mL). The product was a yellow solid (1.59 g, 75% yield);mp, 214.5-216.5° C.; ¹H NMR (DMSO-d₆) δ1.98-2.09 (m, 1H, CHH), 2.49-2.62(m, 2H, CH₂), 2.81-2.95 (m, 1H, CHH), 3.04 (s, 6H, CH₃), 5.08 (dd,J=5.5, 12.7 Hz, 1H, NCH), 7.23 (d, J=6.6 Hz, 1H, Ar), 7.26 (d, J=8.1 Hz,1H, Ar), 7.63 (dd, J=6.9, 8.6 Hz, 1H, Ar), 11.09 (br s, 1H, NH); ¹³C NMR(DMSO-d₆) δ22.10, 30.96, 42.95, 48.77, 112.99, 113.41, 122.59, 133.90,135.22, 149.88, 166.29, 167.13, 170.06, 172.83; Anal Calcd forC₁₅H₁₅N₃O₄: C, 59.80; H, 5.02; N, 13.95. Found: C, 59.60; H, 4.94; N,13.80.

EXAMPLE 9 2-(2,6-Dioxo(3-piperidyl))-4-chloroisoindoline-1,3-dione

2-(2,6-Dioxo(3-piperidyl))-4-chloroisoindoline-1,3-dione was prepared bythe procedure of Example 1 from 3-chlorophthalic anhydride (0.40 g, 2.2mmol), 3-aminopiperidine-2,6-dione hydrogen chloride (0.36 g, 2.2 mmol)and sodium acetate (0.19 g, 2.4 mmol) in acetic acid (10 mL). Theproduct was a white solid (0.44 g, 69% yield); mp, 290.0-291.5° C.; ¹HNMR (DMSO-d₆) δ2.05-2.11 (m, 1H, CHH), 2.49-2.64 (m, 2H, CH₂), 2.64-2.92(m, 1H, CHH), 5.17 (dd, J=5.2, 12.7 Hz, 1H, NCH), 7.86-7.94 (m, 3H, Ar),11.17 (br s, 1H, NH); ¹³C NMR (DMSO-d₆) δ21.83, 30.91, 49.12, 122.41,126.94, 129.84, 133.52, 136.11, 136.39, 164.77, 165.76, 169.73, 172.77;Anal Calcd for C₁₃H₉N₂O₄Cl: C, 53.35; H, 3.10; N, 9.57; Cl, 12.11.Found: C, 53.37; H, 2.94; N, 9.30, Cl, 11.97.

EXAMPLE 104-Methyl-2-(2,6-dioxo-3-methyl-(3-piperidyl))isoindoline-1,3-dione

4-Methyl-2-(2,6-dioxo-3-methyl-(3-piperidyl))isoindoline-1,3-dione wasprepared by the procedure of Example 1 from 3-methylphthalic anhydride(0.27 g, 1.7 mmol), 3-amino-3-methylpiperidine-2,6-dione hydrogenchloride (0.30 g, 1.7 mmol) and sodium acetate (0.15 g, 1.8 mmol) inacetic acid (10 mL). The product was a white solid (0.13 g, 27 % yield);mp, 248.0-250.0° C.; ¹H NMR (DMSO-d₆) δ1.89 (s, 3H, CH₃), 2.01-2.08 (m,1H, CHH), 2.49-2.70 (m, 3H, CHH, CH₂), 2.55 (s, 3H, CH₃), 7.62-7.74 (m,3H, Ar), 10.99 (br s, 1H, NH); ¹³C NMR (DMSO-d₆) δ17.0, 21.0, 28.6,29.1, 58.6, 120.7, 127.5, 131.5, 134.2, 136.8, 137.2, 167.7, 168.6,172.1, 172.3; Anal. Calcd. for C₁₅H₁₄N₂O₄+0.3 H₂O: C, 61.77; H, 5.05; N,9.60. Found: C, 62.05; H, 4.94; N, 9.20.

EXAMPLE 11

Tablets, each containing 50 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline, can be preparedin the following manner:

Constituents (for 1000 tablets) 1-oxo-2-(2,6-dioxo-piperidin- 50.0 g3-yl)-4-methyl-isoindoline lactose 50.7 g wheat starch 7.5 gpolyethylene glycol 6000 5.0 g talc 5.0 g magnesium stearate 1.8 gdemineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. The active ingredient, lactose, talc, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to a boiling solution ofthe polyethylene glycol in 100 mL of water. The resulting paste is addedto the pulverulent substances and the mixture is granulated, ifnecessary with the addition of water. The granulate is dried overnightat 35° C., forced through a sieve of 1.2 mm mesh width and compressed toform tablets of approximately 6 mm diameter which are concave on bothsides.

EXAMPLE 12

Gelatin dry-filled capsules, each containing 100 mg of1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline, can beprepared in the following manner:

Composition (for 1000 capsules) 1,3-dioxo-2-(2,6-dioxo- 100.0 gpiperidin-3-yl)-4-methyl- isoindoline microcrystalline cellulose 30.0 gsodium lauryl sulfate 2.0 g magnesium stearate 8.0 g

The sodium lauryl sulfate is sieved into the1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline through asieve of 0.2 mm mesh width and the two components are intimately mixedfor 10 minutes. The microcrystalline cellulose is then added through asieve of 0.9 mm mesh width and the whole is again intimately mixed for10 minutes. Finally, the magnesium stearate is added through a sieve of0.8 mm width and, after mixing for a further 3 minutes, the mixture isintroduced in portions of 140 mg each into size 0 (elongated) gelatindry-fill capsules.

EXAMPLE 13

A 0.2% injection or infusion solution can be prepared, for example, inthe following manner:

1,3-dioxo-2-(2,6-dioxopiperidin- 5.0 g 3-yl)-4,7-dimethylisoindolinesodium chloride 22.5 g phosphate buffer pH 7.4 300.0 g demineralizedwater to 2500.0 mL

1-Dioxo-2-(2,6-dioxopiperidin-3-yl)-4,7-dimethylisoindoline is dissolvedin 1000 mL of water and filtered through a microfilter. The buffersolution is added and the whole is made up to 2500 mL with water. Toprepare dosage unit forms, portions of 1.0 or 2.5 mL each are introducedinto glass ampoules (each containing respectively 2.0 or 5.0 mg ofimide).

What is claimed is:
 1. A compound selected from the group consisting of(a) a substantially chirally pure (R)-enantiomer, a substantiallychirally pure (S)-enantiomer, or a mixture of the (R)- and(S)-enantiomers of a 2-(2,6-dioxopiperidin-3-yl)-isoindoline of theformula:

in which a first of R¹ and R² is halo, alkyl of from 1 to 4 carbonatoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in which eachalkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl, the second ofR¹ and R², independently of the first, is hydrogen, halo, alkyl of from1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, alkylamino inwhich alkyl is of from 1 to 4 carbon atoms, dialkylamino in which eachalkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl, and R³ ishydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl, and (b) an acidaddition salt of said 2-(2,6-dioxopiperidin-3-yl)-isoindolines whichcontain a nitrogen atom capable of being protonated.
 2. A compoundselected from the group consisting of (a) a substantially chirally pure(R)-enantiomer, a substantially chirally pure (S)-enantiomer, or amixture of the (R)- and (S)-enantiomers of a2-(2,6-dioxopiperidin-3-yl)-isoindoline of the formula:

in which a first of R¹ and R² is halo, alkyl of from 1 to 4 carbonatoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in which eachalkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl, the second ofR¹ and R², independently of the first, is hydrogen, halo, alkyl of from1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, alkylamino inwhich alkyl is of from 1 to 4 carbon atoms, dialkylamino in which eachalkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl, and R³ ishydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl, and (b) an acidaddition salt of said 2-(2,6-dioxopiperidin-3-yl)-isoindolines whichcontain a nitrogen atom capable of being protonated.
 3. The compoundaccording to claim 2 in which R¹ and R³ are hydrogen.
 4. A compoundaccording to claim 3 in which R² is methyl, ethyl, chloro, or methoxy.5. A compound according to claim 2 in which R² and R³ are methyl and R¹is hydrogen .
 6. A pharmaceutical composition comprising in combinationwith a pharmaceutical carrier a quantity of a compound according toclaim 2 sufficient upon administration in a single or multiple doseregimen to reduce levels of inflammatory cytokines in a mammal.
 7. Thecompound according to claim 1 in which R¹ and R³ are hydrogen.
 8. Thecompound according to claim 7 which R² is methyl, ethyl, chloro, ormethoxy.
 9. A compound according to claim 1 in which R² and R³ aremethyl and R¹ is hydrogen.
 10. The compound according to claim 2 whichis a member selected from the group consisting of a substantiallychirally pure(S)-1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline, asubstantially chirally pure(R)-1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline, andmixtures thereof.
 11. The compound according to claim 2 which is amember selected from the group consisting of a substantially chirallypure (S)-1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-ethylisoindoline, asubstantially chirally pure(R)-1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-ethylisoindoline, andmixtures thereof.
 12. The compound according to claim 2 which is amember selected from the group consisting of a substantially chirallypure (S)-1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4,7-dimethylisoindoline,a substantially chirally pure(R)-1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4,7-dimethylisoindoline, andmixtures thereof.
 13. The compound according to claim 2 which is amember selected from the group consisting of a substantially chirallypure(S)-1,3-dioxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4-methylisoindoline,a substantially chirally pure(R)-1,3-dioxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4-methylisoindoline,and mixtures thereof.
 14. The compound according to claim 2 which is amember selected from the group consisting of a substantially chirallypure(S)-1,3-dioxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4,7-dimethylisoindoline,a substantially chirally pure(R)-1,3-dioxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4,7-dimethylisoindoline,and mixtures thereof.
 15. The compound according to claim 1 which is amember selected from the group consisting of a substantially chirallypure (S)-1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline, asubstantially chirally pure(R)-1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline, and mixturesthereof.
 16. The compound according to claim 1 which is a memberselected from the group consisting of a substantially chirally pure(S)-1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-ethylisoindoline, asubstantially chirally pure(R)-1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-ethylisoindoline, and mixturesthereof.
 17. The compound according to claim 1 which is a memberselected from the group consisting of a substantially chirally pure(S)-1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,7-dimethylisoindoline, asubstantially chirally pure(R)-1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,7-dimethylisoindoline, andmixtures thereof.
 18. The compound according to claim 1 which is amember selected from the group consisting of a substantially chirallypure (S)-1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4-methylisoindoline,a substantially chirally pure(R)-1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4-methylisoindoline, andmixtures thereof.
 19. The compound according to claim 1 which is amember selected from the group consisting of a substantially chirallypure(S)-1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4,7-dimethylisoindoline,a substantially chirally pure(R)-1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4,7-dimethylisoindoline,and mixtures thereof.
 20. A pharmaceutical composition comprising incombination with a pharmaceutical carrier a quantity of a compoundaccording to claim 1 sufficient upon administration in a single ormultiple dose regimen to reduce levels of inflammatory cytokines in amammal.